Cardiac glycosideelicited inotropy is thought to be due to inhibition of the Na, KATPase. In the rat, guinea pig, and human hearts, the occupancy of highaffinity as well as lowaffinity ouabain binding sites has been found to correlate with intoropy, and the propportion of high affinity sizes has been found to change in development and in pathological conditions. I previously discovered two distinct isozymes of the Na,KATPase in the brain, and demonstrated that they have markedly different affinites for ouabain. This revised proposal presents evidence that cardiac tissue contains similar, but not identical isozymes of the Na,KATPase. The objective is to identify, characterize, and localize high- and lowaffinity Na,KATPases, either in cardiac cells or in autonomic nerve endings. There has been little structural characterization of the cardiac Na,KATPase, and the molecular identity of its high affinity ouabain site has not been known until now. In contrast, we have purified the Na,KATPases of rat brain axolemma (high affinity) and kidney (low affinity) and established that they have structural and antigenic differences. We now have evidence that suggests that the high affinity form from the heart is antigenically different from that of the brain. Because the isozymes of the Na,KATPase are closely related, very selective probes are needed to distinguish them. We now want to develop specific monoclonal antibodies to the axolemma, kidney, cardiac, and sympathetic neuron isozymes, as molecular tools for the study of their structure and distribution. Monoclonal antibodies that recognize distinct antigens will be obtained with a strategy for supressing the immunological response to shared antigens. The identity of the Na,KATPases in the heart will be determined by detection of specific determinants in their proteolytic peptide maps, and their structures will be compared with those of the axolemma and kidney Na,KATPases. The distribution of the isozymes in cardiac tissue and its sympathetic innervation will be determined by immunocytochemistry. We will evaluate the ouabain affinities of the different Na,KATPases, to provide a basis for predicting their different physiological roles.